Determination of CaffeineIn Beverages: A Review

American Journal of Engineering Research (AJER)

2014

Research Paper

American Journal of Engineering Research (AJER) e-ISSN : 2320-0847 p-ISSN : 2320-0936 Volume-3, Issue-8, pp-124-137

Open Access

Determination of CaffeineIn Beverages: A Review

Igelige Gerald1, David Ebuka Arthur1, Adebiyi Adedayo2.

1Department of Chemistry, Ahmadu Bello University Zaria. 2Sheda Sci. and Tech. complex FCT, PMB 186 Garki Abuja

ABSTRACT :Caffeine is a well-known stimulant which is added as an ingredient to various carbonated soft

drinks. Caffeine has drawn more attention due to its physiological effects beyond that of its stimulatory effect. Consumers are interested in knowing the exact amounts of caffeine existing in beverages. However, limited data exist, especially for store brand beverages. Therefore, it is pertinent to review the various methods that will effectively determine the caffeine contents in different carbonated drinks. HPLC, UV-Visible Spectrometry and Gas Chromatography are among the popular used methods.

KEYWORDS;Carbonated-drinks, Analysis, Extraction, Additive, Determination, Caffeine.

I. INTRODUCTION

Caffeine is a naturally occurring alkaloid which is found in the leaves, seeds and fruits of over 63 plants species worldwide (Abdul Muminet al., 2006; NourVioletaet al.,2008; Wanyikaet al., 2010; VioletaNouret al., 2010) It is an alkaloid of methylxanthine family (Wanyikaet al.,2010; Marcia et al., 2002). The methylxanthines caffeine (1,3,7-trimethyxanthine), theobromine (3,7- dimethylxanthine), and theophylline (1,3-dimethylxanthine) can be normally found in cola nuts, coffee beans, cocoa beans, tea leaves, mate leaves and other kinds of plants (Paradkar and Irudayaraj, 2002). While coffee and tea beverages naturally contain caffeine and other methylxanthines, caffeine serves as an ingredient in many carbonated soft drinks including colas, pepper-type beverages, and citrus beverages. Pure caffeine occurs as odorless, white, fleecy masses, glistening needles of powder. Its molecular weight is 194.19g, melting point is 236?C, point at which caffeine sublimes is 178?C at atmospheric pressure, pH is 6.9 (1% solution), specific gravity is 1.2, volatility is 0.5%, vapor pressure is 760mmHg at 178?C, solubility in water is 2.17%, vapor density 6.7 (Komeset al., 2009; NourVioletaet al., 2008; Hiroshi Ashiharaet al., 1996; Abdul Muminet al., 2006).Caffeine has drawn more attention in the past decades due to its physiological effects beyond that of its stimulatory effect. The Food and Drug Administration (FDA) defines caffeine as a generally recognized as safe (GRAS) substance. However, FDA specifies that the maximum amount in carbonated beverages is limited to 0.02% (FDA 2006). Therefore, the highest legal amount of caffeine allowed in a 355 mL (12oz) can of soft drink is about 71mg. Caffeine has attracted the interest of consumers and health professionals alike due to its wide consumption in the diet by a large percentage of the population and its pharmacological effects in humans (Mandel 2002). The human's saliva caffeine level, which demonstrates the extent of absorption, peaks around 40 minutes after caffeine consumption (Liguoriet al 1997). Its physiological effects on many body systems have been reported by researchers, including the central nervous, cardiovascular, gastrointestinal, respiratory, and renal systems (Nehliget al 1992). The International Olympic Committee (IOC) defined caffeine as a drug and abuse is indicated when athletes have urine caffeine concentrations higher than 12g/mL (de Aragaoet al 2005).

II. CAFFEINE CHEMISTRY AND GENERAL INFORMATION

Caffeine (1,3,7-trimethyxanthine), theophylline (3,7- dimethylxanthine), and theobromine (1,3dimethylxanthine) are in the family of alkaloid methylxanthines.



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Caffeine (1, 3, 7-trimethylxanthine)

Caffeine is an odorless, white solid that has the form of needles or powder. Caffeine has a bitter taste. The molar mass of caffeine is 194.19 g/mol. Caffeine is slightly soluble in water due to its moderate polarity. Caffeine is a natural central nervous system stimulant, having the effects of reducing drowsiness and recovering alertness. Since it is widely consumed by humans, caffeine is considered the most frequently used psychoactive substance in the world (Ligouriet al 1997).

Physiological effects of caffeine to human Caffeine has numerous physiological effects on major organ systems, including the nervous system,

cardiovascular system, digestive system, and respiratory system. Renal function and skeletal muscles are also affected by caffeine. Numerous studies have proven caffeine to be a stimulant to human's central nervous system (Spiller, 1998).It is also increase heartbeat rate, dilate blood vessels and elevate levels of free fatty acids and glucose in plasma. 1 g of caffeine leads to insomnia, nervousness, nausea, ear ringing, flashing of light derillum and tremulosness. In cases of overdosing and in combination with alcohol, narcotics and some other drugs, these compounds produce a toxic effect, sometimes with lethal outcome (Mamina and Pershin, 2002; Ben Yuhas, 2002; Wanyikaet al., 2010; James et al., 1990; Tavallali and Sheikhaei, 2009). Caffeine facilitates the conduction velocity in the heart and directly affects the contractility of the heart and blood vessels. Nevertheless, caffeine may significantly reduce cerebral blood flow by constricting of cerebral blood vessels. Caffeine provides a diuretic effect due to elevating the blood flow and glomerular filtration rate of the kidneys. Heartburn is an issue for some subjects' gastrointestinal system after consuming caffeine. The effects of caffeine to skeletal muscles are mainly the increasing occurrence of tremors (James 1991; Spiller 1998).

Relevant Literatures Many methods exist for determining the methylxanthine contents of food and beverages. Some of these

methods include UV-Visible spectrophotometry, potentiometry, high performance liquid chromatography (HPLC), ion chromatography, high performance thin layer chromatography (HPTLC), capillary electrophoresis, micellar capillary electrophoresis, gas chromatography, and solid-phase microextraction gas chromatography (Armenta et al., 2005). Of the above methods, HPLC has become one of the most commonly used analytical methods.One study demonstrated using an HPLC method with an octadecylsilyl (ODS) column and a wateracetonitrile-phosphoric acid mobile phase to analyze eight catechins and caffeine. Within 20 min, the catechins (epicatechin, epigallocatechin, epicatechingallate, epigallocatechingallate, catechin, catechingallate, gallocatechin and gallocatechingallate) and caffeine were separated by an acetonitrile gradient. Two different types of Japanese green teas, Matcha and Sencha, both high and low grades for each tea, had their catechins and caffeine contents determined. The researchers found the caffeine contents were higher in Matcha tea than in Sencha tea (Gotoet al., 1996). Wang et al., (2000) applied an isocratic elution system to determine the contents of catechins, caffeine, and gallic acid in green and black tea. The separation system included a C18 reversephase column, a mobile phase of methanol/water/orthophosphoric acid (20/79.9/0.1), and an UV detector. The flow rate was set at 1.0 mL/min. The wavelength of detection was 210 nm. The validation of this method was confirmed by all analytes exhibiting good linearity within the range tested and correlation coefficients ranging from 0.988 to 1.000. The amounts of caffeine in Gunpower, roasted green tea (RGT), Sencha, Keemun, and Sri Lanka were found to be 23.9, 30.3, 28.9, 38.2, and 22.9 mg/100 mL, respectively (Wang et al., 2000).



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Mashkouriet al., (2003) quantitated the caffeine existing in black tea leaves by Fourier transform infrared

(FTIR) spectrometry. The caffeine of tea samples was extracted using CHCl after wetting with an aqueous NH

3

3

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solution. The spectrometric data were collected over the wave number range of 1800-1300 cm . This 11 method

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had a detection limit of 35g/mL, a sampling frequency of 6 h , and a coefficient of variation of 0.8%. A black

tea sample contained 3.68% w/w caffeine. The authors obtained similar results for the caffeine content from

FTIR (3.68 ? 0.03% w/w) and a reference HPLC technique (3.60 ? 0.07% w/w). The advantages of the FTIR

method for determining caffeine in tea leaves includes its quickness, precision, and accuracy, enabling it to be a

possible alternative to the HPLC method (Mashkouriet al., 2003). However, one potential shortcoming of this

method is the fairly high detection limit.

Nishitani and Sagesaka (2004) developed an improved HPLC analytical method for simultaneously determining caffeine and the eight catechins as well as other phenolic compounds in tea. The proposed method provided additional ability to analyze phenolic compounds when compared with former HPLC methods. This procedure was based on an improved reverse-phase ODS column operated at 4?C, a binary gradient elution system of water-methanol-ethylacetate-phosphoric acid, and a photodiode array detector. The quantitative measurement of eight catechins and caffeine confirmed the validity of this proposed method. The detection limits of these analytes ranged from 1.4-3.5 ng per injection volume. The recovery rates of the analyses were in the range of 96-103%. The caffeine contents of Sencha, Matcha, Gunpowder, Tie Kuan yin, and Darjeeling determined in this study were 2.94?0.007, 3.62?0.005, 2.61?0.059, 2.51?0.019, and 3.24?0.016% (dry weight), respectively (Nishitani and Sagesaka 2004). Caudle et al., (2001) tried to improve the Association of Official Analytical Chemists (AOAC) official analytical method for analyzing methylxanthines in cocoa-based food products. Theobromine and caffeine contents could be obtained by 12 reverse-phase HPLC. The AOAC method's degree of accuracy and precision was not reliable, especially for caffeine. In this study, the AOAC analytical method only showed recoveries of theobromine and caffeine to be 89.3 and 74.5%, respectively. The authors successfully changed from an organic extraction to an aqueous extraction and analyzed the samples via reverse-phase HPLC to improve the recoveries of theobrominethe samples via reverse-phase HPLC to improve the recoveries of theobromine and caffeine to 99.6 and 103.4%, respectively (Caudle et al.,2001).

Zuoet al., (2002) analyzed various substances in several green, Oolong, black and pu-erh teas by HPLC. They used a methanol-acetate-water buffer gradient elution system and a C-18 column; detection utilized a photodiode array detector. After multiple extractions with aqueous methanol and acidic methanol solutions, four major catechins, gallic acid and caffeine could be simultaneously determined within 20 min. This improved the previous studies' problem of catechins and caffeine remaining in tea residues after a single extraction. The results demonstrated that green teas contain higher amounts of catechins than Oolong, pu-erh, and black teas due to their fermentation processes reducing the levels of catechins significantly. An interesting finding was a lower caffeine content in Oolong teas, especially in Fujian Oolong tea (Zuoet al., 2002).

Horieet al., (1997) adapted capillary zone electrophoresis (CZE) in order to simultaneously determine the major compounds in green tea. Separation occurred in a fused-silica capillary column. The borax buffer was set at pH 8.0, and UV detection was at 200 nm. The major compounds in green tea were epicatechin, epigallocatechin, epicatechingallate, epigallocatechingallate, catechin, caffeine, theanine, and ascorbic acid. The authors found the concentration of each compound was significantly different among each tea sample. One interesting finding was relatively lower caffeine contents in canned tea drinks. The authors concluded CZE is more appropriate for analyzing the properties and contents of green tea than HPLC due to its shorter analysis time and ability to separate more compounds (Horieet al., 1997). Schulz et al., (1999) investigated the accuracy of rapidly predicting the amounts of polyphenol and alkaloid compounds in the leaves of green tea by a nearinfrared reflectance spectroscopic (NIRS) method. The pretreatment of the NIR spectra with weighted multiple scatter correction effectively eliminated interferences of scatter and improved the final calibration results. The results were compared with those from analysis by HPLC. The potential of this NIRS method is demonstrated by the high correlation between its prediction and HPLC values for caffeine and major catechins. The authors claimed that the NIRS method may be an alternative technique to HPLC due to its high degree of accuracy for prediction and analysis time of less than 1 min per measurement (Schulz et al., 1999). Farah et al., (2006) investigated the relationship between the Arabica coffee cup quality and the contents of sucrose, caffeine, trigonelline, and chlorogenic acids. The researchers applied reverse-phase HPLC analysis to determine each compound. Sucrose was analyzed by using 80% acetonitrile and 20% water as the mobile phase and a refractive index detector. For analyzing caffeine, the UV detector was set at 272 nm. The mobile phase was composed of



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60% water and 40% methanol. The results demonstrated that the caffeine content was the highest in the highest

quality sample and the lowest content was found in the poorest quality sample. However, trigonelline and 3,4-

dicaffeoylquinic acid gave a better indication of high quality coffee (Farah et al., 2006). Huck et al., (2005)

compared the contents of caffeine, theobromine, and theophylline in 83 liquid coffee extracts determined by a

NIRS method and HPLC coupled to mass spectrometry method. In the NIRS method, the spectra were recorded

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over a wave number range of 4008 to 9996 cm with a resolution of 12 cm in the reflectance mode. The

authors obtained high robustness and reproducibility of the NIRS model for quantification of caffeine and

theobromine. The lower limit of detection made it difficult for theophylline to fit the NIRS model and correctly

be determined. Nevertheless, NIRS provides the coffee industry with an alternative method to quickly determine

caffeine and theobromine (Huck et al., 2005).

Chen and Wang (2001) analyzed the level of artificial sweeteners (sodium saccharin, aspartame, acesulfame-K), preservatives (benzoic acid, sorbic acid), caffeine, theobromine, and theophylline in carbonated cola drinks, fruit juice drink, fermented milk drink, preserved fruit, and one pharmaceutical preparation by an ion exchange chromatography method. Analytes were separated using an anion-exchange analytical column maintained at 40?C and detected by wavelength-switching ultraviolet absorption. The detection limits ranged from 4-30 ng/mL for all analytes. The average recoveries for samples ranged from 85 to 104%. In addition, the data obtained from this method were in good agreement with those determined by reference HPLC procedures. Two carbonated cola drinks were found to contain around 36 mg caffeine/12 oz (Chen and Wang 2001). Chen et al. (2006) investigated the feasibility of using near infrared (NIR) spectroscopy as a fast method which is nondestructive and less time consuming thanother frequently used analytical methods for estimating the content of caffeine and total polyphenols in green tea. The calibration was performed by a partial least squares (PLS) algorithm. The result indicated that correlation coefficients of the prediction models were approximately 0.97 for the caffeine and 0.93 for total polyphenols. This method's potential to rapidly determine the caffeine and polyphenols of tea to control industrial processes has been proven by this study (Chen et al., 2006).

Yao et al. (2006) examined 20 leaf tea and 36 teabag samples obtained from Australian supermarkets. Each sample was prepared as a diluted tea solution, which was treated with lead acetate and hydrochloric acid solutions. After filtering and treating with a sulfuric acid solution, the measurement of caffeine was completed by using a UV/Visible spectrophotometer at 570 nm. The results showed that caffeine contents of black leaf tea and teabags were 3.89 and 3.87%, respectively. Similar results were found in the green leaf tea and teabags, 3.71 and 3.83%, respectively. These contents are generally higher than that claimed by the manufacturers (i.e., < 3%). This study revealed a need to establish quality control for both imported and Australian-made teas (Yao et al., 2006). Brunettoet al. (2007) developed a reversed-phase HPLC method with an on-line sample cleanup to determine theobromine, theophylline, and caffeine in cocoa samples. The cocoa samples were prepared by an on-line solid-phase extraction of analytes and loaded into a home-made dry-packed precolumn with ODS-C18 in a column-switching system. The mobile phase consisted of 20% of methanol in water, under isocratic conditions, at a flow-rate of 1.4 mL/min. Chromatographic separation was performed on a NOVA-PAK C18 column (150 mm x 3.9 mm, 4 m). The procedure demonstrated a recovery of over 95% with coefficients of variation less than 3.2%. The precolumn proved its long average life span by showing no signs of deterioration after approximately 1000 injections of sample cocoa extracts (Brunettoet al., 2007).

Pura (2001) modified a HPLC method for determining caffeine and theobromine contents in aqueous cocoa extracts. Instead of directly injecting the extracts on the column, the improved method can successfully remove the interfering cocoa pigments by passing them through a Sep-pak C18 cartridge which was also used to separate the theobromine and caffeine. This method enhanced the efficiency of the column and prolonged its life. After this treatment, the recoveries of caffeine and theobromine were 98.0-100.1 and 97.8-100%, respectively. The modified method displayed good resolution and sharp peaks on chromatograms that favored correct determination of theobromine and caffeine (Pura, 2001). Thomas et al. (2004) measured the contents of caffeine, theobromine, and theophylline in a food-matrix standard reference material (SRM) 2384, Baking Chocolate by a reverse-phase HPLC method. The stationary phase was composed of an inactive silica support to which C-18 was bonded. The mobile phase consisted of 10% acetronitrile90% water (pH acidified to 2.5 with acetic acid). The flow rate was at 1.5 mL/min and UV detection was at 274 nm. The results of each sample could be obtained within 15 min. The results showed the reproducibility for caffeine, theobromine, and theophylline determinations was 5.1, 2.3, and 1.9%, respectively. This method had a limit of determination for all analytes at levels less than 100 ng/mL or 0.1g/mL. The measurements of caffeine, theobromine, and theophylline of SRM 2384



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BakingChocolate were comparable with those from National Institute of Standard and Technology (Thomas et al., 2004). Abourashed and Mossa (2004) applied HPTLC densitometric analysis to determine the level of caffeine in several herbal products and energy drinks. The HPTLC plates were made of pre-coated silica gel. The solvent system contained 85% ethyl acetate and 15% methanol. The wavelength for detecting caffeine was set at 275 nm. The proposed method had a mean recovery of 98.9 ? 3.5% with a coefficient of variation less than 5%. The caffeine ranges of herbal products and energy drinks in this study were found at 4.76-13.29% (w/w) and 0.011-0.032% (w/w), respectively. The HPTLC method demonstrated effective determination of caffeine for stimulant herbal products and carbonated energy drinks (Abourashed and Mossa 2004). Armenta et al. (2005) applied a solid-phase Fourier transform-Raman (SP-FT-Raman) spectrometry-based method to determine caffeine contents in commercial energy drinks. The caffeine content of each sample was obtained

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from setting Raman intensity between 573 and 542 cm with a two points corrected baseline between 580 and

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540 cm . The limit of detection of SP-FT-Raman method was 18 g/mL. The combination of FT-Raman and solid-phase increased the sensitivity of detecting caffeine by a factor of 31 times when compared with using direct Raman measurement alone. The results of caffeine contents obtained from SP-FT-Raman method and liquid chromatography (LC) found no significant differences between the two methods. The SP-FT-Raman method displayed higher sampling frequency than the LC method. However, the LC method had a lower detection limit (0.05 g/mL). The reduced reagent consumption and waste generation are also benefits of this method as compared to the LC method (Armenta et al., 2005).

Lucenaet al. (2005) manipulated a continuous flow auto analyzer for sequential determination of total sugars, class IV caramel and caffeine contents in 20 different soft drink samples. This apparatus consisted of online coupling of a continuous solid-phase extraction unit and two detectors which were UV-visible and evaporative light scattering (ELSD) detectors. The caffeine has the property of being retained on the sorbent column and other compounds can be preferentially determined due to their low affinity to the sorbent column. The caffeine can be detected later by the ELSD after it has been eluted with acetonitrile and the signal registered in the ELSD. In order to evaluate the performance of this analyzer, the authors carried out a recovery test. The results ranged from 90 to 102%. Unspecified colas were found to contain caffeine ranging from 14.9 mg/12 oz to 49.7 mg/12 oz (Lucenaet al., 2005). Walker et al. (1997) utilized capillary electrophoresis (CE) to simultaneously analyze the aspartame, benzoic acid, and caffeine contents of carbonated beverages in 2 min with 20 mM glycine buffer at pH 9.0 and detection at 215 nm. Good reproducibility for both peak area and migration times were observed (2.0-3.8% and 0.13-0.37%, respectively). The spiked recovery of the analytes ranged from 98 to 114%. The results of soft drinks samples in this study were comparable with those data evaluated by HPLC, but slightly higher in some cases using CE. The main advantages of CE over HPLC are relatively simpler operation, lower cost, no organic mobile solvents, and a shorter analysis time (Walker et al., 1997).

Types of drinks :Non-alcoholic soft drink beverage can be divided into fruit drinks and soft drinks. Soft drinks can be divided into carbonated and non-carbonated drinks. Examples of carbonated drinks are Cola, lemon and oranges and non-carbonated drinks include mango drinks. Soft drinks can also be divided into cola products and non-cola products. Cola products like Pepsi, Coca-Cola, Thumps Up, and Diet Coke, Diet Pepsi etc. account for nearly 61-62% of the total soft drinks market. Non-Cola products constitute 36%, and based on the types of flavors available can be divided into Orange, Cloudy Lime, Clear Lime and Mango (India Infoline Sector Report, 2002).Below are highlight of some work which employed the various methods in analyzing the level of caffeine from different beverages.In this review, I will attempt to go in details into some of the experiments carried out in determining caffeine content and also publishing of some of their results as shown below;

The quantitative determination of caffeine in beverages and soft drinks using UV wavelength spectroscopy JENWAY, producersof instrumentations for Chemistry related practical, sampled five beverages and soft drink. Samples were chosen they include instant coffee (Nescafe), brewed tea (PG Pyramid Tea Bags), Coca Cola, Pepsi Cola and Red Bull. The analysis is performed on a Jenway 7305 spectrophotometer controlled using the free-of charge PC software, supplied with each model in the 73 series. The software allows the user to emulate all measurement tasks normally performed on the instrument with the additional benefit of allowing data to be seamlessly transferred to external Microsoft office applications. TheReagents used are Caffeine,Dichloromethane and Purified water



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